Gérard Potron

Human Blood Monocytes Interact with Type I Collagen Through αxβ2 Integrin (CD11c-CD18, gp150-95)

Human blood monocytes are attracted into connective tissues during early steps of inflammation and wound healing, and locally interact with resident cells and extracellular matrix proteins. We studied the effects of type I collagen on monocyte adhesion and superoxide anion production, using human monocytes elutriated from peripheral blood and type I collagen obtained from rat tail tendon. Both acid-soluble and pepsin-digested type I collagens promoted the adhesion of monocytes, whereas only acid-soluble collagen with intact telopeptides induced the production of superoxide. Adhesion and activation of monocytes on acid-soluble type I collagen depended on the presence of divalent cations. mAbs directed against integrin subunits CD11c and CD18 specifically inhibited adhesion and activation of monocytes on type I collagen. Protein membrane extracts obtained from monocytes were submitted to affinity chromatography on collagen I-Sepharose 4B, and analyzed by Western blotting using specific anti-integrin subunit Abs. In the case of both acid-soluble and pepsin-digested collagens, two bands were revealed with mAbs against CD11c and CD18 integrin subunits. Our results demonstrate that monocytes interact with type I collagen through CD11c-CD18 (αxβ2) integrins, which promote their adhesion and activation. For monocyte activation, specific domains of the type I collagen telopeptides are necessary. Interactions between monocytes and collagen are most likely involved in the cascade of events that characterize the initial phases of inflammation.

Inhibition of fMLP-triggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptor.

Adenosine (Ado) is a potent anti‐inflammatory agent acting on a variety of cell functions. However, its effects on human monocytes have been less well characterized. We investigated the effect of Ado and its receptor‐specific analogs on NADPH oxidase activity with the use of luminol‐enhanced chemiluminescence (CL). Adenosine inhibited fMLP‐triggered NADPH oxidase activity with a maximal inhibition of 55 ± 5%. IB‐MECA, a selective A3 Ado receptor agonist reduced fMLP triggered NADPH oxidase activity more potently than the A2 receptor agonist CGS 2180 HCl (CGS) and the A1 Ado receptor agonist N‐2‐phenylethyladenosine (R‐PIA). The inhibitory effect of Ado was reversed by neither the A1 Ado receptor antagonist 1,3‐dipropyl‐8(2‐amino‐4chlorophenyl)‐xanthine (PACPX) nor the A2 Ado receptor antagonist 3,7‐dimethyl‐1‐(2‐propynyl)xanthine (DMPX). It was significantly reversed by the A1/A3 Ado receptor antagonist xanthine amine congener (XAC). Pretreatment of monocytes by cytochalasin B reversed the effect of Ado but not of dibutyryl cAMP (dBcAMP) on fMLP‐CL response. KT 5720, a specific cAMP‐dependent protein kinase inhibitor completely counteracted the inhibition of NADPH oxidase activity by dBcAMP but not by Ado. Using flow cytometry, we observed that Ado did not inhibit intracellular oxidative metabolism, whereas dBcAMP did. Furthermore, the inhibition of NADPH oxidase activity by Ado was not mediated by changes in cytosolic calcium. These results demonstrated that Ado inhibited NADPH oxidase activity via A3 Ado receptor independently of cAMP elevation or changes in calcium mobilization. J. Leukoc. Biol. 66: 495–501; 1999.

FACTOR V LEIDEN : DETECTION IN WHOLE BLOOD BY ASA PCR USING AN ADDITIONAL MISMATCH IN ANTEPENULTIMATE POSITION

Factor V Leiden mutation was initially detected in thrombophilic patients and relatives by PCR RFLP (Restriction Fragment Length Polymorphism) according to Bertina (1). This technique presents some drawbacks and the current trend is to simplify the diagnosis. We describe a technique of Allele Specific Amplification (ASA) which is optimized in terms of reliability: an additional mismatch in antepenultimate position enables to obtain the same specificity as PCR RFLP. Furthermore, coamplification of internal control warrants an optimal sensitivity. All the PCR have been simplified: the DNA extraction improvement allows to analyse the genotype with only a few microliters of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4 degrees C for several days). This technique saves time. Moreover, full automation of the ASA technique may be shortened thanks to the lack of extraction and the positive/negative reading of the PCR signal.

Thromboprophylaxis in Hip Fracture Surgery: A Pilot Study Comparing Danaparoid, Enoxaparin and Dalteparin

A pilot study was performed to compare the thromboprophylactic effect of danaparoid, enoxaparin and dalteparin in patients with hip fracture. The study was a prospective, randomised assessor-blind, four-centre trial. Prophylaxis was given for 9–11 days, whereafter bilateral phlebography was performed. A total of 197 patients were randomised. There were no statistically significant differences in the frequency of deep vein thrombosis, in blood loss or bleeding complications between the three prophylaxis groups. In conclusion, this moderately sized study revealed no statistically significant difference in efficacy or safety between danaparoid, enoxaparin and dalteparin in patients undergoing hip fracture surgery.

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures.

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three‐dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three‐dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen‐contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein‐zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non‐specific inhibitors of serine proteinases, and by η‐amino‐caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva.

Coexpression of tissue factor and tissue factor pathway inhibitor by human monocytes purified by leukapheresis and elutriation.Response of nonadherent cells to lipopolysaccharide

BACKGROUND: Counterflow centrifugal elutriation is the method of choice for obtaining a large quantity of highly purified monocytes. In spite of the fact that this technique has been used for many years to isolate monocytes for cellular immunotherapy, it is not known whether the process of elutriation can stimulate tissue factor (TF) expression and therefore trigger coagulation in patients receiving these cell preparations. The aim of the present study is thus to identify TF and TF pathway inhibitor (TFPI) in elutriated monocytes and to evaluate their ability to trigger thrombin generation.

Identification of chromosomal loci associated with non‐P‐glycoprotein–mediated multidrug resistance to topoisomerase II inhibitor in lung adenocarcinoma cell line by comparative genomic hybridization

In order to identify genomic changes associated with an etoposide resistance acquisition, we used comparative genomic hybridization (CGH) to compare a human lung adenocarcinoma cell line, A549 wild type, and three sublines, A549‐VP1‐3, exposed to increasing concentrations of the topoisomerase II inhibitor, VP16. R‐banding karyotype, fluorescence in situ hybridization (FISH), and Southern blot for the MLL gene were also performed. The CGH analysis showed that the A549‐VP3 cell line shared chemoresistance‐specific abnormalities (amplification of 11q23–qter, loss of chromosome 17, and deletions of 2p14–pter and 2q23–q24). FISH analysis confirmed the loss of one chromosome 17 in the three resistant sublines and revealed an increased fragmentation of chromosome 2 in more than two segments, depending on the etoposide concentration. FISH with an MLL gene probe showed additional signals of MLL (from three in the A549‐WT to seven in the A549‐VP3 cell line) translocated onto several other chromosomes. Southern blot indicated an amplification of the MLL gene, dependent on the etoposide concentration, without gene rearrangement. The CGH results are suggestive of loci that could be associated with the acquisition of an etoposide‐chemoresistant phenotype. Deletion of the 2p region has already been reported, without any candidate gene being identified. The role of MLL in leukemogenesis has previously been demonstrated, but its role in the development of other tumors or its significance in the chemoresistance process remains to be elucidated.

Cytogenetic characterization of chromosomal rearrangement in a human vinblastine-resistant CEM cell line: use of comparative genomic hybridization and fluorescence in situ hybridization.

In order to identify genomic changes associated with drug-resistance acquisition, we performed R-banding karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization to compare a human T-cell lymphoblastic leukemia cell line, CEM-wild type, and a subline with resistance to vinblastine (CEM-VLB) and overexpressing P-glycoprotein. Comparative genomic hybridization analysis showed that the CEM-VLB cell line carried chemoresistance-associated chromosomal abnormalities (amplification of 7q11 approximately q22, losses of chromosomes 2, 3, 5, 9, 10, and 16, and deletion of 4q13 approximately qter). Fluorescence in situ hybridization identified an amplified 7q21 region translocated on the short arm of a chromosome 2. This region contained the MDR1 gene locus and probably neighboring genes, such as SRI or MDR3/ABCB4. According to previous reports, this chromosomal rearrangement occurred during drug selection and attested a resistance acquisition.

Mechanisms of the platelet aggregation induced by activated neutrophils and inhibitory effect of specific PAF receptor antagonists

The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed platelets in human. It potentiates platelet aggregation induced by agonists in platelet rich plasma as well as in whole blood. This activation involves the phosphoinositide metabolism. Specific PAF receptor antagonists gingkolides (BN 50726, BN 52021, BN 54068, BN 54062, BN 50730, BN 50749, BN 50744) and benzodiazepine Web2086 antagonize this neutrophil-induced platelet aggregation. BN 50,730, BN 50,749 and Web 2086 can fully inhibit this aggregation at the final concentration of 10(-6) M. Preincubation of platelets with synthetic PAF also inhibits this activation through a desensitization of the receptor. These data suggest the major involvement in our model of PAF acether in the platelet-neutrophil interactions.

Separation of dendritic cells from highly purified human monocytes by counterflow centrifugation induces tissue factor expression

BACKGROUND: In vitro generation of dendritic cells (DCs) from human monocytes represents a promising tool in immunotherapy. However, it is not known whether the separation of DCs from monocytes induces tissue factor expression and therefore may trigger coagulation in patients receiving these DC preparations. The aim of this study is thus to analyze tissue factor expression on monocyte‐derived DCs and to compare their ability to trigger thrombin generation to that of macrophages obtained from the same monocytes.